Targeting Gαi/s Proteins with Peptidyl Nucleotide Exchange Modulators.

Chemical probes that specifically modulate the activity of heterotrimeric G proteins provide excellent tools for investigating G protein-mediated cell signaling. Herein, we report a family of selective peptidyl Gαi/s modulators derived from peptide library screening and optimization. Conjugation to a cell-penetrating peptide rendered the peptides cell-permeable and biologically active in cell-based assays. The peptides exhibit potent guanine-nucleotide exchange modulator-like activity toward Gαi and Gαs. Molecular docking and dynamic simulations revealed the molecular basis of the protein-ligand interactions and their effects on GDP binding. This study demonstrates the feasibility of developing direct Gαi/s modulators and provides a novel chemical probe for investigating cell signaling through GPCRs/G proteins.

Csk, a critical link of g protein signals to actin cytoskeletal reorganization.

Heterotrimeric G proteins can signal to reorganize the actin cytoskeleton, but the mechanism is unclear. Here we report that, in tyrosine kinase Csk-deficient mouse embryonic fibroblast cells, G protein (Gbetagamma, Galpha(12), Galpha(13), and Galpha(q))-induced, and G protein-coupled receptor-induced, actin stress fiber formation was completely blocked. Reintroduction of Csk into Csk-deficent cells restored the G protein-induced actin stress fiber formation. Chemical rescue experiments with catalytic mutants of Csk demonstrated that the catalytic activity of Csk was required for this process. Furthermore, we uncovered that Gbetagamma can both translocate Csk to the plasma membrane and directly increase Csk kinase activity. Our genetic and biochemical studies demonstrate that Csk plays a critical role in mediating G protein signals to actin cytoskeletal reorganization.

G protein betagamma subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry.

The nervous system can modulate neurotransmitter release by neurotransmitter activation of heterotrimeric GTP-binding protein (G protein)-coupled receptors. We found that microinjection of G protein betagamma subunits (Gbetagamma) mimics serotonin's inhibitory effect on neurotransmission. Release of free Gbetagamma was critical for this effect because a Gbetagamma scavenger blocked serotonin's effect. Gbetagamma had no effect on fast, action potential-evoked intracellular Ca2+ release that triggered neurotransmission. Inhibition of neurotransmitter release by serotonin was still seen after blockade of all classical Gbetagamma effector pathways. Thus, Gbetagamma blocked neurotransmitter release downstream of Ca2+ entry and may directly target the exocytotic fusion machinery at the presynaptic terminal.

Coupling Gbetagamma-dependent activation to channel opening via pore elements in inwardly rectifying potassium channels.

G protein-coupled inwardly rectifying potassium channels, GIRK/Kir3.x, are gated by the Gbetagamma subunits of the G protein. The molecular mechanism of gating was investigated by employing a novel yeast-based random mutagenesis approach that selected for channel mutants that are active in the absence of Gbetagamma. Mutations in TM2 were found that mimicked the Gbetagamma-activated state. The activity of these channel mutants was independent of receptor stimulation and of the availability of heterologously expressed Gbetagamma subunits but depended on PtdIns(4,5)P(2). The results suggest that the TM2 region plays a key role in channel gating following Gbetagamma binding in a phospholipid-dependent manner. This mechanism of gating in inwardly rectifying K+ channels may be similar to the involvement of the homologous region in prokaryotic KcsA potassium channel and, thus, suggests evolutionary conservation of the gating structure.

Modulation of glycine-activated ion channel function by G-protein betagamma subunits.

Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor. Overexpression of three distinct G protein alpha subunits (Galpha), as well as the Gbetagamma scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gbetagamma increased the GlyR open probability (nP(o)). Our results indicate that this interaction of Gbetagamma with GlyRs regulates both motor and sensory functions in the central nervous system.

Endothelial G protein beta-subunits trigger nitric oxide-but not endothelium-derived hyperpolarizing factor-dependent dilation in rabbit resistance arteries.

A single subtype of heterotrimeric G protein-coupled receptor controls both nitric oxide (NO) (sensitive to L-arginine analogues) and endothelium-derived hyperpolarizing factor (EDHF) (sensitive to high-external K(+) and apamine) production by the vascular endothelium leading to dilation. We hypothesized that alpha- and betagamma-subunits of the G protein serve as distinct intermediates to produce NO and EDHF. In pressurized resistance arteries, selective pinocytotic endothelial incorporation of specific antibodies (Abs) directed against alpha(q/11)-subunits abolished acetylcholine (Ach)-mediated dilation but failed to influence oxymetazoline (Oxy, alpha(2)-adrenergic receptor agonist)-induced dilation. In contrast, alpha(i1-2)-subunit Abs prevented Oxy- but not Ach-induced dilation. Thus, as expected, endothelial muscarinic and alpha(2)-adrenoceptors couple to G(q) protein and G(i) proteins, respectively. beta-subunit Abs reduced both Ach- and Oxy-induced dilation. The beta-subunit Abs abolished the nitro-L-arginine (L-NNA)-sensitive component but did not impair the high-external K(+)-sensitive component of the dilation induced by Ach and Oxy. Thus, G protein beta-subunits primarily accounted for NO production. Neutralization of Hsp90 and inhibition of the phospholipase C by U73122 (1 micromol/L) or intracellular Ca(2+) buffering with BAPTA-AM (10 micromol/L) sharply reduced NO-dependent but not K(+)-sensitive dilation. In conclusion, mobilization of the G protein beta-subunit is pivotal to NO-dependent dilation triggered through muscarinic and alpha(2)-adrenergic receptors. In contrast, receptor-operated EDHF-dependent dilation was insensitive to beta-subunit Abs. Although not directly activating the NO pathway, alpha-subunit activation is an absolute prerequisite for receptor-operated endothelium-dependent dilation of resistance arteries.

Syntaxin 1A supports voltage-dependent inhibition of alpha1B Ca2+ channels by Gbetagamma in chick sensory neurons.

N-type Ca(2+) channels are modulated by a variety of G-protein-coupled pathways. Some pathways produce a transient, voltage-dependent (VD) inhibition of N channel function and involve direct binding of G-protein subunits; others require the activation of intermediate enzymes and produce a longer-lasting, voltage-independent (VI) form of inhibition. The ratio of VD:VI inhibition differs significantly among cell types, suggesting that the two forms of inhibition play unique physiological roles in the nervous system. In this study, we explored mechanisms capable of altering the balance of VD and VI inhibition in chick dorsal root ganglion neurons. We report that (1) VD:VI inhibition is critically dependent on the Gbetagamma concentration, with VI inhibition dominant at low Gbetagamma concentrations, and (2) syntaxin-1A (but not syntaxin-1B) shifts the ratio in favor of VD inhibition by potentiating the VD effects of Gbetagamma. Variations in expression levels of G-proteins and/or syntaxin provide the means to alter over a wide range both the extent and the rate of Ca(2+) influx through N channels.

Investigation of the role of the carboxyl-terminal tails of the alpha and beta isoforms of the human thromboxane A(2) receptor (TP) in mediating receptor:effector coupling.

We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A(2) receptor (TP) to Galpha(16) and Galpha(12) members of the G(q) and G(12) families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP(Delta328) cells which over-express a variant of TP truncated at the point of divergence of TPalpha and TPbeta, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha(16) to affect increases in inositol 1,4,5-trisphosphate (IP(3)) and mobilisation of intracellular calcium ([Ca(2+)](i)) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca(2+)](i) mobilisation in cells co-transfected with Galpha(12), neither receptor generated corresponding increases in IP(3), indicating that the Galpha(12)-mediated increases in [Ca(2+)](i) do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca(2+) channels, reduced [Ca(2+)](i) mobilisation in TPalpha and TPbeta cells co-transfected with Galpha(12) to approximately 40% of that mobilised in its absence, whereas [8-(N,N-diethylamino)-octyl-3,4, 5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca(2+) release, had no effect on [Ca(2+)](i) mobilisation by either receptor isoform co-transfected with Galpha(12). Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP(Delta328) signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha(11), Galpha(12) or Galpha(16) subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP(Delta328) coupled to Galpha(s), leading to increased adenosine 3',5'-cyclic monophosphate (cAMP), rather than to Galpha(i). Whereas TP(Delta328) signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha, co-transfection of Galpha(s) did not augment cAMP generation by TP(Delta328). Hence, from these studies involving the wild type TPalpha, TPbeta and TP(Delta328), we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha(11) and Galpha(16) members of the G(q) family or to Galpha(12); it may play a role in determining G(s) versus G(i) coupling and may act as a determinant of coupling efficiency.

GTP-binding protein Gq mediates muscarinic-receptor-induced inhibition of the inwardly rectifying potassium channel IRK1 (Kir 2.1).

The inwardly rectifying potassium channel IRK1, a member of the Kir 2.0 family, is inhibited by m1 muscarinic receptor stimulation. In this study the mechanism of action underlying the muscarinic response was investigated by identification of the subtype of heterotrimeric G-protein involved in transduction of the signal. tsA201 cells were simultaneously transfected with cDNAs encoding IRK1, m1 and the Galpha subunit of either G(q), G(12) or G(13). The whole-cell patch-clamp technique was used to study the effects of G-protein transfection. Antibodies generated against the C-terminal regions of Galpha(q/11) and Galpha(12) were used to confirm G-protein expression by Western blot. When challenged with carbachol, IRK1 currents recorded from cells co-transfected with Galpha(q) were potently inhibited compared with controls. Conversely, co-transfection with Galpha(12) or Galpha(13) subunits had no effect on muscarinic-receptor-induced inhibition of IRK1. Concentration response curves revealed that carbachol was 16 times more potent at inhibiting IRK1 currents in cells co-transfected with Galpha(q) as compared with Galpha(12) co-transfected cells. Immunoblotting illustrated low levels of endogenous Galpha(q/11) and Galpha(12) in untransfected tsA cells. Transfection with Galpha(q) or Galpha(12) cDNAs greatly increased the levels of G-protein expression in both cell populations. G-protein expression did not interfere with m1 muscarinic receptor expression levels. These findings suggest that the m1 muscarinic-receptor-induced inhibition of IRK1 is mediated by the heterotrimeric G-protein, Galpha(q), in tsA cells.

Pharmacological comparison of a recombinant CB1 cannabinoid receptor with its G(alpha 16) fusion product.

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB(1) agonist-mediated signal transduction via an engineered G(alpha 16) system is different than that of the G(i/o) coupling normally preferred by the CB(1) receptor, we transfected the human recombinant CB(1) receptor (hCB(1)) or a fusion protein comprising the hCB(1) receptor and G(alpha 16) (hCB(1)-G(alpha 16)) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB(1)-G(alpha 16) fusion protein was found to be similar to that for hCB(1): HU 210 > CP 55,940 > or = SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [(35)S]GTP gamma S binding or inhibited forskolin-stimulated cAMP, presumably by coupling to G(i/o), in cells expressing hCB(1) but not hCB(1)-G(alpha 16). However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCB(1)-G(alpha 16) but not hCB(1). These data demonstrate that ligand affinities for the hCB(1) receptor are not affected by fusion to the G(alpha 16) subunit. Furthermore, there is essentially no difference in the function of the hCB(1) receptor when coupled to G(i/o) or G (alpha 16).

Feeding induced by food deprivation is differentially reduced by G-protein alpha-subunit antisense probes in rats.

Antisense oligodeoxynucleotide (AS ODN) probes directed against the alpha-subunit of different G-proteins have been used to differentiate feeding responses in rats elicited by different opioid agonists, including morphine, beta-endorphin and dynorphin. Furthermore, antisense probes directed against G(o)alpha, but not G(s)alpha, G(q)alpha or G(i)alpha, significantly reduced nocturnal feeding in rats. The present study examined whether food intake and weight changes elicited by 24 h of food deprivation were significantly altered by ventricular administration of antisense probes directed against either G(i)alpha(1), G(i)alpha(2), G(i)alpha(3), G(s)alpha, G(o)alpha, G(q)alpha or G(x/z)alpha as well as a control nonsense probe in rats. Deprivation-induced weight loss was significantly enhanced by antisense probes directed against G(s)alpha and G(x/z)alpha, whereas weight recovery 24 h following reintroduction of food was significantly reduced by antisense probes directed against G(i)alpha(2), G(q)alpha and G(o)alpha. Selective antisense probe effects were noted for deprivation-induced intake with G(s)alpha and G(q)alpha probes exerting the greatest reductions, G(x/z)alpha, G(i)alpha(2), and G(i)alpha(3) probes exerting lesser effects, and G(i)alpha(1) and G(o)alpha probes failing to affect deprivation-induced intake. Importantly, the nonsense control probe failed to alter deprivation-induced intake or weight. The reductions in deprivation-induced intake by AS ODN probes directed against G(s)alpha or G(q)alpha were not accompanied by any evidence of a conditioned taste aversion. These data indicate important distinctions between G-protein mediation of different effector signaling pathways mediating feeding responses elicited under natural (e.g. nocturnal feeding) and regulatory challenge (e.g. food deprivation) conditions.

Direct modulation of phospholipase D activity by Gbetagamma.

Phospholipase D-mediated hydrolysis of phosphatidylcholine is stimulated by protein kinase C and the monomeric G proteins Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of this enzyme. Using purified proteins, we have identified a novel inhibitor of phospholipase D activity, Gbetagamma subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Galpha and Gbetagamma subunits, allowing subsequent interaction with distinct effectors. Gbeta1gamma1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gbeta1gamma1 and Gbeta1gamma2 inhibited stimulated phospholipase D1 activity in a dosedependent manner in reconstitution assays. Reconstitution assays suggest this interaction occurs through the amino terminus of phospholipase D, because Gbeta1gamma1 is unable to inhibit an amino-terminally truncated phospholipase D construct, PLD1.d311, which like full-length phospholipase D isoforms, requires phosphatidylinositol-4,5-bisphosphate for activity. Furthermore, a truncated protein consisting of the amino-terminal region of phospholipase D containing the phox/pleckstrin homology domains was found to interact with Gbeta1gamma1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gbeta1gamma2 was also found to inhibit phospholipase D activity under basal and stimulated conditions in MDA-MB-231 cells, which natively express both phospholipase D1 and phospholipase D2. These data demonstrate that Gbetagamma directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.

Phospholipase Cbeta2 binds to and inhibits phospholipase Cdelta1.

Phospholipase Cbeta (PLCbeta) isoforms, which are under the control of Galphaq and Gbetagamma subunits, generate Ca2+ signals induced by a broad array of extracellular agonists, whereas PLCdelta isoforms depend on a rise in cytosolic Ca2+ for their activation. Here we find that PLCbeta2 binds strongly to PLCdelta1 and inhibits its catalytic activity in vitro and in living cells. In vitro, this PLC complex can be disrupted by increasing concentrations of free Gbetagamma subunits. Such competition has consequences for signaling, because in HEK293 cells PLCbeta2 suppresses elevated basal [Ca2+] and inositol phosphates levels and the sustained agonist-induced elevation of Ca2+ levels caused by PLCdelta1. Also, expression of both PLCs results in a synergistic release of [Ca2+] upon stimulation in A10 cells. These results support a model in which PLCbeta2 suppresses the basal catalytic activity of PLCdelta1, which is relieved by binding of Gbetagamma subunits to PLCbeta2 allowing for amplified calcium signals.

Conformational analysis of the Galpha(s) protein C-terminal region.

The C-terminal domain of the heterotrimeric G protein a-subunits plays a key role in selective activation of G proteins by their cognate receptors. Several C-terminal fragments of Galpha(s) (from 11 to 21 residues) were recently synthesized. The ability of these peptides to stimulate agonist binding was found to be related to their size. Galpha(s)(380-394) is a 15-mer peptide of intermediate length among those synthesized and tested that displays a biological activity surprisingly weak compared with that of the corresponding 21-mer peptide, shown to be the most active. In the present investigation, Galpha(s)(380-394) was subjected to a conformational NMR analysis in a fluorinated isotropic environment. An NMR structure, calculated on the basis of the data derived from conventional 1D and 2D homonuclear experiments, shows that the C-terminal residues of Galpha(s)(380-394) are involved in a helical arrangement whose length is comparable to that of the most active 21 -mer peptide. A comparative structural refinement of the NMR structures of Galpha(s)(380-394) and Galpha(s)(374-394)C379A was performed using molecular dynamics calculations. The results give structural elements to interpret the role played by both the backbone conformation and the side chain arrangement in determining the activity of the G protein C-terminal fragments. The orientation of the side chains allows the peptides to assume contacts crucial for the G protein/receptor interaction. In the 15-mer peptide the lack as well as the disorder of some N-terminal residues could explain the low biological activity observed.

The amino terminus of Galpha(z) is required for receptor recognition, whereas its alpha4/beta6 loop is essential for inhibition of adenylyl cyclase.

G(z) couples to most of the known G(i)-linked receptors and its alpha subunit (Galpha(z)) inhibits adenylyl cyclases as efficiently as Galpha(i) subtypes. A series of chimeric Galpha subunits with different portions of Galpha(z) and Galpha(t1) (a regulator of cGMP phosphodiesterase) were constructed to study the essential structural elements of Galpha(z) that determine receptor coupling and effector interaction. The receptor-mediated functions of the chimeras were assessed in two aspects: 1) stimulation of type 2 adenylyl cyclase through the release of betagamma subunits from the chimeras, and 2) inhibition of isoproterenol-stimulated adenylyl cyclase by the chimeric Galpha subunits. The results suggested that the presence of both termini of Galpha(z) were critical for coupling to delta-opioid receptor, with the N-terminal region being more important. Moreover, a stretch of amino acids (295-319) corresponding to the alpha4/beta6 loop was identified as one of the adenylyl cyclase inhibitory domains of Galpha(z).

Activated G alpha q inhibits p110 alpha phosphatidylinositol 3-kinase and Akt.

Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Galphaq(Q209L) mutant to explore the effects of Galphaq activation on signaling through the PI3K/Akt pathway. Transient expression of Galphaq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Galphaq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Galphaq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Galphaq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110alpha (but not p110beta) immunoprecipitates. Receptor activation of Galphaq also selectively inhibited PI3K activity in p110alpha immunoprecipitates. Active Galphaq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Galphaq(Q209L) co-immunoprecipitated with the p110alpha-p85alpha PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Galphaq(Q209L) with purified recombinant p110alpha-p85alpha in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Galphaq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Galphaq and p110alpha PI3K.

Molecular mechanisms for the activation of voltage-independent Ca2+ channels by endothelin-1 in chinese hamster ovary cells stably expressing human endothelin(A) receptors.

We demonstrated recently that in Chinese hamster ovary cells stably expressing human recombinant endothelin(A) receptors (CHO-ET(A)R), endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC), which can be distinguished by Ca(2+) channel blockers such as 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate (LOE 908). We also reported that CHO-ET(A)R couples with G12 in addition to G(q) and G(s). The purpose of the present study was to identify the G proteins involved in the activation of these Ca2+ channels by ET-1, using mutated ET(A)Rs with coupling to either G(q) or G(s)/G12 (designated ET(A)RDelta385 and SerET(A)R, respectively) and a dominant-negative mutant of G12 (G12G228A). ET(A)RDelta385 is truncated immediately downstream of Cys385 in the C terminus as palmitoylation sites, whereas SerET(A)R is unpalmitoylated because of substitution of all the cysteine residues to serine (Cys383Cys385-388 --> Ser383Ser385-388). In CHO-ET(A)RDelta385, stimulation with ET-1 activated only SOCC. In CHO-SerET(A)R or CHO-ET(A)R pretreated with U73122, an inhibitor of phospholipase C (PLC), ET-1 activated only NSCC-1. Dibutyryl cAMP alone did not activate any Ca2+ channels in the resting and ET-1-stimulated CHO-SerET(A)R. Microinjection of G12G228A abolished the activation of NSCC-1 and NSCC-2 in CHO-ET(A)R and that of NSCC-1 in CHO-SerET(A)R. These results indicate that ET(A)R activates three types of Ca2+ channels via different G protein-related pathways. NSCC-1 is activated via a G12-dependent pathway, NSCC-2 via G(q)/PLC- and G12-dependent pathways, and SOCC via a G(q)/PLC-dependent pathway.

Neurite outgrowth induced by cyclic AMP can be modulated by the alpha subunit of Go.

Although abundant Go has been found in nervous tissues and it has been implicated in neuronal differentiation, the mechanism of how Go modulates neuronal differentiation has not been defined. Here, we report that the alpha subunit of Go (alphao) modulates neurite outgrowth by interfering with the signaling pathway initiated by cyclic AMP (cAMP). In F11 cells, cAMP induced neurite outgrowth and activated cAMP-responsive element binding protein (CREB). Specific inhibition of cAMP-dependent protein kinase reduced both CREB activity and neurite outgrowth (NOG). Interestingly, cAMP reduced phosphorylation of extracellular signal-regulated kinase (Erk). Neither a dominant negative form nor an active form of Ras altered neurite outgrowth. Expression of alphao (alphao(wt)) decreased the average length of neurites but increased the number of neurites per cell. An active mutant, alphaoQ205L, which lost GTPase activity and thus could not bind to Gbetagamma, gave similar results, suggesting that the effect of alphao is not mediated through Gbetagamma. Expression of ao(wt) or alphaoQ205L also prohibited CREB activation. Thus, activation of Erk may not be essential for neuronal differentiation in F11 cells and alphao may cause changes in NOG by inhibiting CREB activation.

Direct interactions between the heterotrimeric G protein subunit G beta 5 and the G protein gamma subunit-like domain-containing regulator of G protein signaling 11: gain of function of cyan fluorescent protein-tagged G gamma 3.

We used fluorescence resonance energy transfer imaging of enhanced cyan fluorescent protein (CFP)-tagged and enhanced yellow fluorescent protein (YFP)-tagged protein pairs to examine the hypothesis that G protein gamma subunit-like (GGL) domain-containing regulators of G protein signaling (RGS) can directly bind to the Gbeta5 subunit of heterotrimeric G proteins in vivo. We observed that Gbeta5 could interact with Ggamma2 and Ggamma13, after their expression in human embryonic kidney 293 cells. Interestingly, although untagged Ggamma3 did not interact with Gbeta5, CFP-tagged Ggamma3 strongly interacted with YFP-tagged Gbeta5 in FRET studies. Moreover, CFP-Ggamma3 supported Ca(2+) channel inhibition when paired with Gbeta5 or YFP-Gbeta5, indicating a "gain of function" for CFP-Ggamma3. Gbeta5 could also interact with RGS11 and its N-terminal, but not its C-terminal domain. On the other hand, RGS11 did not interact with Gbeta1. These studies demonstrate that the GGL domain-containing N terminus of RGS 11 can directly interact with Gbeta5 in vivo and supports the hypothesis that this interaction may contribute to the specificity of Gbeta5 interactions with cellular effector molecules.

The effect of protein kinase C activation on G(z)-mediated regulation of type 2 and 6 adenylyl cyclases.

Three serine-to-alanine mutants of the alpha subunit of the heterotrimeric G protein G(z) (alpha(z)) were examined for their signaling properties in the presence of phorbol ester treatment. All three alpha(z) mutants resembled wild-type alpha(z) in their abilities to inhibit alpha(s)-stimulated type 6 adenylyl cyclase (AC6) and phorbol ester treatment reduced their magnitudes of inhibition. Depending on the permissive condition, the betagamma-mediated stimulation of type 2 adenylyl cyclase (AC2) was differentially regulated by alpha(z) and the three mutants. Mutation of Ser(27) but not Ser(16) of alpha(z) affected the efficient release of betagamma subunits upon receptor activation and abolished the stimulation of phosphorylated but not alpha(s)-stimulated AC2.